snoRNAs are highly conserved non-coding RNAs that direct two types of site-specific post-transcriptional modifications of other RNAs - pseudouridinylation and 2'-O-methylation. Dysregulation of specific snoRNAs is associated with increased risk for breast, prostate, lung, brain and colorectal cancers as well as leukemia and multiple myeloma, but the mechanistic connections between altered snoRNA function and cancer are not understood. Recent discoveries herald a paradigm shift in the snoRNA field. First, snoRNAs were shown to have a greatly expanded list of potential RNA targets, including mRNAs. Second, some snoRNA-directed modifications were shown to be regulated by environmental changes. Finally, the presence of pseudouridine was shown to change how the genetic code is interpreted by ribosomes. Together, these results suggest potentially widespread regulatory functions for snoRNAs through directing post-transcriptional modification of mRNAs. We hypothesize that snoRNAs direct physiologically relevant pseudouridinylation of mRNA targets by a mechanism that involves relaxed specificity pairing between guide sequence and target RNA. To test this hypothesis, we have developed an efficient method for mapping the sites of snoRNA-directed pseudouridinylation genome-wide with single nucleotide precision (Pseudo-seq). Our preliminary results from Pseudo-seq experiments provide the first evidence that endogenous mRNAs are pseudouridinylated. Thus, we are uniquely well positioned to investigate this new paradigm for snoRNA function. In Aim 1 we will identify the regulated targets of snoRNA-directed pseudouridinylation in response to nutrient deprivation, oxidative stress and heat shock using Pseudo-seq. Mutants in the catalytic component of snoRNA enzymes, Cbf5/Dyskerin, will be used to determine which pseudouridines are formed by a snoRNA-dependent mechanism. Completion of this aim will comprehensively identify mRNA targets of regulated snoRNA-dependent modification in yeast. Aim 2 will use computational and targeted genetic approaches to dissect the sequence requirements for regulation by snoRNAs. Completion of this aim will facilitate accurate prediction of snoRNA targets in all eukaryotic organisms. Finally, Aim 3 will use mass spectroscopy to determine the functional consequences of mRNA pseudouridinylation for decoding by the ribosome. Completion of this aim has the potential to reveal substantial rewiring of the genetic code. Human health relevance: The results of this study will reveal new biological functions of snoRNAs and thereby illuminate the potential mechanisms by which snoRNA dysfunction leads to cancer. We believe that in the future, a better understanding of the function of snoRNAs in cells may lead to better treatment of cancers associated with perturbation of the function of snoRNA genes.